Opium alkaloid antigens and antibodies specific therefor

ABSTRACT

Opium alkaloid antigens are prepared by coupling opium alkaloid haptens to immunogenic carrier materials. In preferred embodiments proteins are used as carrier materials and the coupling is effected through the carboxyl group of a carboxy lower alkyl derivative of the phenolic hydroxy group of said alkaloid and a free amino group on the protein thereby yielding a covalent peptide bond. The resulting antigens produce immunological effects when injected into host animals, including the formation of opium alkaloid specific antibodies. These specific antibodies are useful in bioanolytical techniques for the assay of opium alkaloids in biological fluids.

United States tel Spector 1 Jan.9,1973

[ OPIUM ALKALOID ANTIGENS AND ANTIBODIES SPECIFIC THEREFOR [5 6]References Cited UNITED STATES PATENTS 11/1942 Fell ..140/57 3/1945 Fell5/1933 Pitkin ..424/96 FOREIGN PATENTS OR APPLICATIONS 116,806 9/1899Germany OTHER PUBLICATIONS Journal of Immunology, 38, pp. 479-490 (1940)Hooker et a1.

Atti and Accad. Italia, Rend. Classe Sci. 2 (7), pp..19311.LQIlPALLMiIIQ M Chem. Abstracts, Vol.29, 193s,'0'i'7 8016 MaScherpa. Chem. Abstracts, V01. 61, 1964, 9914 Kramarenko. Chem.Abstracts, Vol. 73, 1970, 5416d, Spector et al.

Primary Examiner-l-loward E. Schain Attorney-Samuel L. Welt, Jon S.Saxe, Bernard S. Leon, Ronald A. Schapira and William H. Epstein [57]ABSTRACT Opium alkaloid antigens are prepared by coupling opium alkaloidhaptens to immunogenic carrier materials. In preferred embodimentsproteins are used as carrier materials and the coupling is effectedthrough the carboxyl group of a carboxy lower alkyl derivative of thephenolic hydroxy group of said alkaloid and a free amino group on theprotein thereby yielding a covalent peptide bond. The resulting antigensproduce immunological effects when injected into host animals, includingthe formation of opium alkaloid specific antibodies. These specificantibodies are useful in bioanolytical techniques for the assay of opiumalkaloids in biological fluids.

5 Claims, No Drawings OPIUM ALKALOID ANTIGENS AND ANTIBODIES SPECIFICTHEREFOR RELATED APPLICATIONS The present application is acontinuation-in-part of copending application Ser. No. 36,999, filed May13, 1970 now abandoned.

BACKGROUND OF THE INVENTION The large increase in the use of narcoticagents including the opium alkaloids by the general population hasbrought with it a substantial need to improve analytical techniques forthe determination of such agents in biological fluids. In many instancesmedical treatment centers are faced with the need of determining theidentity of a narcotic agent taken by a patient who, being in a comatosecondition, is unable to supply such information to the treatingphysician. At present procedures for the identification of opiumalkaloids involve extraction and thin-layer chromatographic methods.These techniques have the disadvantage of being relatively timeconsuming, laborious and lacking great-sensitivity. A more rapid andhighly sensitive assay for the presence of opium alkaloids in biologicalfluids would thus represent an extremely important advance in the art.

It has been known for some time that various small molecules (haptens),which by themselves are wholly devoid of antigenicity, can modify theantigenic properties of a protein when the small molecule is combinedwith the protein through stable covalent linkages. Thus, U.S. Pat. No.2,372,066 discloses that antigens may be prepared by combininghistamines or histamine-like compounds by linking the imidazole ring toa desired protein through a radical containing a group capable ofcoupling with the protein. These antigens are used either by directinjection into a subject whereby resistance, refractoriness or activeimmunity is developed in said subject or for injecting into host animalsfrom which antibodies specific to the hapten moiety, e.g., the histamineor histamine-like substance, are developed. Similarly, Landsteiner in anarticle entitled Specificity of Serological Reactions, HarvardUniversity Press, Cambridge, Massachusetts (1945) disclosed theutilization of p-amino-benzene arsonic acid as a hapten which whencoupled to a protein was able to produce specific antibodies in a hostanimal.

SUMMARY OF THE INVENTION The present invention relates to a novel classof an tigens comprising an opium alkaloid hapten moiety coupled to animmunogenic carrier-material. In preferred embodiments the opiumalkaloid is covalently bonded to a protein molecule through a carboxylower alkyl linking group via a peptide linkage. This peptide linkageinvolves the amino group of the protein molecule and the carboxy groupof the carboxy lower alkyl linking group. This linking group is bondedto the opium alkaloid as a derivative of the phenolic hydroxyl group.Additionally, the present invention relates to antibodies which willcomplex with some specificity to the opium alkaloids. These antibodiesare produced by treating host animals with the aforesaid antigens. Suchspecific antibodies are readily isolated from sera obtained from hostanimals after treatment of these host animals with the aforesaidantigen.

The term lower alkyl as used above is meant to include straight andbranched chain saturated hydrocarbon radicals having from one to sixcarbon atoms such as methyl, ethyl, propyl and the like.

The opium alkaloid of particular preference in the practice of thepresent invention is morphine. Other opium alkaloids useful in thisinvention include, for ex ample, heroin and codeine and derivativesthereof which do not involve the phenolic hydroxy group. It has beenfound that the antibodies elicited by the utilization of a morphinehapten-protein antigen are specific not only to morphine but alsoclosely related derivatives such as codeine and to a lesser extentnalorphine.

As used herein the term carrier material is meant to include thosematerials which have the property of independently eliciting animmunogenic response in a host animal when injected therein and whichcan be coupled by covalent bonding to said opium alkaloid hapten.Suitable carrier materials include, for example, materials such asproteins; natural or synthetic polymeric compounds such as polypeptides,e.g., polylysine; polysaccharides; and the like. A particularlypreferred carrier material for the practice of the present invention isprotein.

The identity of the protein carrier material utilized in the preparationof the preferred antigen of the present invention is not critical.Examples of preferred proteins useful in the practice of this inventioninclude the serum proteins preferably mammalian serum proteins,

such as for example, human gamma globulin, human serum albumin, rabbitserum albumin, bovine gamma globulin and bovine serum albumin. Othersuitable protein products will be suggested to one skilled in the art.It is generally preferred that proteins be utilized which are foreign tothe animal host in which the resulting antigen will be employed.

The first step in the preparation of the preferred antigen of thepresent invention involves converting the opium alkaloid to the carboxylower alkyl derivative of the alkaloids phenolic hydroxy group. This ismost conveniently accomplished by reacting the free base of the alkaloidwith an alkali salt of a halo lower (e.g., oneseven carbon atoms,preferably one-four carbon atoms) alkanoic acid. Suitable halo loweralkanoic acids for this purpose include B-halo acetic acid, 7-halopropionic acid, S-halobutyric acid and the like.

The alkali salts useful in the practice of this invention of theaforesaid halo alkanoic acids include the sodium, potassium and lithiumsalts; the sodium salt being of greatest preference. Halo derivatives ofthe aforesaid alkanoic acids include the chloro, bromo, iodo and fluoroderivatives. In a most preferred embodiment of the present inventionmorphine free base is converted to its 3-O-carboxymethyl derivative byreaction with sodium B-chloro acetate. The aforesaid general reaction isfacilitated by conducting it in the presence of a suitable inert organicsolvent such as a lower alkanol, e.g., ethanol. Most preferably thesolvent system is anhydrous and thus an absolute lower alkanol isemploycd. For purposes of the present invention, the term lower alkanolis meant to include straight and branched chain alkanols having from oneto seven carbon atoms.

The reaction introducing the carboxy lower alkyl group into the opiumalkaloid molecule can conveniently be conducted at a temperature in therange of from about room temperature to about C; although higher orlower temperatures may be employed if desired. In preferred embodimentsroom temperature is employed for this reaction.

The coupling of the carboxy lower alkyl derivative of the opium alkaloidwith a protein to form the preferred antigen of the present inventioncan be readily accomplished utilizing techniques now well known inprotein chemistry for establishing peptide bonds. Thus, for example, onesuch technique would involve dissolving the protein and a water solublecoupling agent in water followed by adding a large molar excess of thedesired carboxy lower alkyl opium alkaloid derivative. This reaction maybe conducted at a temperature in the range of from about C. to about50C., although higher or lower temperatures may be employed depending onthe nature of the reactants and the denaturization temperature of theprotein. A most preferable temperature is at about room temperature. Itis desirable to utilize a slightly acidic reaction medium, e.g., amedium having a pH in the range of from about 3 to 6.5, most preferablyin the range of from about 4 to 6.5, e.g., 5.5. Suitable water solublecoupling agents for use in the present invention include water solublecarbodiimides such as, for example, 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Upon completion of the above coupling reaction, the excess haptenmolecules and coupling agents may be removed by dialysis. The dialysismay be monitored by checking the dialysate for the presence of hapten orcoupling agents or, alternatively, may be conducted for a pro-determinedperiod of time, c.g., seven days. The dialysis is conducted in distilledwater which is preferably changed four to five times per day. Purifiedantigen is recovered as a residue in the dialysis bag.

Besides water, other suitable solvent media for use in the abovereaction include, for example, 015 M salt solution (NaCl), a 0.l5 M saltsolution buffered with 0.01 M phosphate buffer (pH 7.4) or a 0.01 Mphosphate buffer solution. The reaction mixture is brought to thedesired pH range by the addition of dilute aqueous acid, e.g., lN HCl.Solvents which denature proteins, e.g., organic solvents such asalcohols, ethers, etc. or strong inorganic acids or bases such asmineral acids or alkali hydroxides should generally not be employed.

The amount of molar excess of the hapten over the protein in theaforesaid reaction will, of course, depend on the identity of the opiumalkaloid derivative utilized and the protein selected for the reaction.Generally, a molar excess in the range of from about 30 to 10,000, mostpreferably in the range of from about 100 to 1,000 of the hapten inrelation to the protein, will be utilized.

It is generally found that from about 3 to about 7 opium alkaloidderivative groups are added to a molecule of protein depending of courseon the amount of molar excess of hapten used. For example, three to fourcarboxymethylmorphine groups will be added to a molecule of bovine serumalbumin (assuming a molecular weight of 70,000 for the protein) when a100 molar excess of the hapten is used.

It is believed that the reaction sequence used in the preparation of theantigen of the present invention can be illustrated by the followingreaction scheme. in such scheme 3-0-carboxy-methylmorphine is utilizedas the exemplar hapten group.

l EtOH llllLiL'J'll where X is halo, M is an alkali metal and n is aninteger from I to 6.

The antigen of the present invention may then be utilized to induceformation of opium alkaloid specific antibodies in the serum of hostanimals by injecting the antigen in such host repeatedly over a periodof time, collecting the serum, precipitating the antibody with a neutralsalt solution and, if desired, purifying the antibody by dialysis andcolumn chromatography. Suitable host animals for this purpose includemammals such as rabbits, horses, goats, guinea pigs, rats, cows, sheep,etc. The resulting antibody will have a multiplicity of active siteswhich will selectively complex with opium alkaloids, the opium alkaloidantigen described above or closely related derivatives.

The formation of opium alkaloid specific antibodies in the host animalsmay be monitored by taking blood samples from the host animal and addingto it an amount of the hapten-protein antigen. The presence ofprecipitate indicates antibody activity. The antigen treatment of theanimal can be continued until the antibody titre reaches a desired levelof activity. For the purposes of this application the antibody titre isdefined as being the maximum concentration of protein precipitatedfollowing the addition of varying known concentrations of antigens tofixed volumes of serum, e.g., 0.5 ml.

The antibodies of the present invention can be isolated from the sera oftreated host animals by utilizing techniques well known in thebiochemical arts. For example, the sera obtained from treating hostanimals can be acted upon by a neutral salt which will effectprecipitation of the desired specific antibodies. Suitable neutral saltsfor this purpose include sodium sulfate, magnesium sulfate, a sodiumhydrogen phosphate mixture of ammonium sulfate. The neutral saltpreferred for the purpose of the present invention is ammonium sulfate.Purification techniques subsequent to the precipitation mixture may alsobe employed such as dialysis and column chromatography. The resultingantibody may be characterized as being a gamma globulin having amolecular weight of about 160,000. This antibody will complex with ahigh degree of specificity with opium alkaloid haptens and antigensderived therefrom.

The specific antibodies of the present invention are useful as reagentsin biochemical assays for the determination of the presence of opiumalkaloids and closely related compounds in biological fluids. Aparticularly preferred assay procedure is an immunoprecipitationprocedure which can be used to measure nanogram amounts of opiumalkaloid, e.g., morphine in serum. in such procedure a known amount oflabeled opium alkaloid is mixed with opium alkaloid antibody and asample containing the unknown quantity of opium alkaloids. The amount ofopium alkaloid in the sample can be determined by measuring the amountof competitive inhibition observed between the binding of the labeledopium alkaloid and the sample opium alkaloid with the opium alkaloidspecific antibody and then calculating the amount of opium alkaloid inthe sample from a standard curve. Suitable labeled opium alkaloids forthis purpose include the isotopically labeled opium alkaloidsparticularly tritiated opium alkaloids, i.c., dihydromorphine-H andmorphine-I as well as opium alkaloids labeled with an electron spinresonance group. Examples of the use of various electron spin resonancelabeled molecules in bioassays are to be found in U.S. Pat. Nos.3,453,288, 3,481,952 and 3,507,876. A preferred radioimmunoprecipitationtechnique for use in the assay of opium alkaloids is described infurther detail in the accompanying examples.

This invention is further illustrated by the following specificexamples.

EXAMPLE 1 Preparation of antigen Morphine was converted to3-0-carboxymethyl morphine by reaction of the alkaloid free base withsodium ,Bchloroacetate in absolute ethanol according to the proceduresdescribed in Houben Fortschritte der Heilstoffchemie, 1, 882 (1901) andBeilstein, Band 27, l56. The derivative product after recrystallizationfrom hot absolute ethanol had a melting point of 292293 C. The3-0-carboxymethyl morphine acid was Dragendorff positive and Paulynegative and had a Rf of 0.6 on thin-layer silica gel chromatographyusing glacial acetic acidzmethanol (1:1) as the solvent system. In thesame solvent system morphine had a Rf of 1.0. The carboxymethyl morphinewas coupled to bovine serum albumin (BSA) in aqueous solution, in thepresence of water soluble carbodiimide by the following procedure. Atotal of 8 mg. of carboxymethyl morphine was dis solved in 2 ml. ofdistilled water containing 10 mg. of BSA. The pH of the mixture wasadjusted to 5.5 with molecule of BSA (assuming a molecular weight of70,000 for the protein).

EXAMPLE 2 Preparation of antibody Rabbits of the New Zealand albinostrain were immunized with 1 mg. of carboxymethyl morphine-BSA. Theimmunogen was dissolved in phosphate buffer saline pH 7.4, emulsifiedwith an equal volume of complete Freunds adjuvant and injected into thefootpad (0.4 ml./footpad). Booster injections of 100 pg. of antigen inadjuvant were given every 6-8 weeks in the footpads and sides.

Antiserum was collected 5-) days after booster injections. Bloodcollected by cardiac puncture was incubated at 37C. for 1 hour and thenkept overnight at 4C. After centrifugation at 5,000 rpm. for 30 minutesat 4C., serum was separated from the clot.

EXAMPLE 3 Radioimmuno assay The radioimmuno assay was performed byincubating various dilutions of antisera obtained in Example 2 in thepresence of dihydromorphine-H (New England nuclear, 388 mc/mM, 100picomoles (4,000 cpm) at 4C. overnight. After incubation a neutral,saturated ammonium sulfate solution (volume equal to incubation medium)was added to all tubes. The precipitate, sedimented by centrifugation at5,000 rpm for 15 minutes at 4C. was washed twice in 50 percent ammoniumsulfate solution. The wash precipitates, containing antibody-boundmorphine, was dissolved in 0.5 ml. of commercial detergent solubilizersuch as NCS solubilizer and quantitatively transferred and counted in aPackard Tri-Carb Liquid Scintillation Spectrometer. The tube whichcontained radioactive dihydromorphine and antiserum but no unlabeledmorphine served as a measure of maximum anti-bodybound radioactivity.The addition of increasing amounts of unlabeled morphine to a fixedamount of dihydro-l-l and antiserum resulted in a competitive inhibition of the labeled dihydromorphine for the formation of theantibody-hapten complex. The data ob tained is summarized below in TableI.

TABLE I Nanograms Percent Inhibition of Binding Non-radioactive MorphineAdded of Dihydromorphine-H The above data clearly demonstrates thesensitivity of the method. When plotted in graphic form the datacontained in the above table demonstrates a linear relationship betweenthe amount of non-radioactive morphine added and the percent ofinhibition found. With the addition of 0.5 nanogram of unlabeledmorphine per tube [a concentration of l nanogram per ml. before addingammonium sulfate] percent of the labeled dihydromorphine was displacedfrom the antibody. The specificity of the antiserum for morphine wasdemonstrated by incubating the labeled hapten with normal rabbit serum.The radioactivity remaining in a precipitate from normal rabbit serumafter washing was slightly above background and was subtracted fromvalues obtained on all other tubes.

Comparison runs were also carried out with other alkaloids. Theinhibitory capacity of codeine, which is morphine-3-methyl ether wascompared with that of morphine. Codeine was found even more effectivethan morphine (on a molar basis) in producing 50 percent inhibition ofprecipitation of radioactivity. This is not surprising since codeinepossesses greater structural similarity to the immunizingcarboxymethylmorphine group than does morphine itself. It was furtherfound that 500 nanograms of nalorphine decreases precipitation ofradioactivity by about 35 percent whereas the same amount of methadonproduced little or no inhibition.

lclairn:

I. An antigen consisting essentially of a carboxy lower alkyl derivativeof the phenolic hydroxy group of an opium alkaloid bonded through apeptide linkage to an amino group of a protein.

2. The antigen of claim 1 wherein said opium alkaloid derivative is3-0-carboxymethylmorphine.

3. The antigen of claim 1 wherein said protein is bovine serum albumin.

4. The antigen of claim 3 wherein three to seven opium alkaloidderivative groups are bonded per molecule of bovine serum albumin.

5. An antigen consisting essentially of a carboxy lower alkyl derivativeof the phenolic hydroxyl group of an opium alkaloid bonded through apeptide linkage to an amino group of an immunogenic carrier materialselected from the group consisting of proteins and polypeptides.

UNITED STATES PATENT OFFICE Certificate Patent No. 3,709,868 PatentedJanuary 9, 1973 Sidney Spector Application having been made by SidneySpector, the inventor named in the patent above identified, andHofimann-La Roche Inc., Nutley, New Jersey, a corporation of New Jersey,the assignee, for the issuance of a certificate under the provision ofTitle 35 Section 256, of the United States Code, adding the name ofCharles W. Parker as a oint inventor, and a showing and proof of factssatisfying the requirements of the said section having been submitted,it is this 5th day of June 1973, certified that the name of the saidCharles W. Parker is hereby added to the said patent as a joint inventorwith the said Sidney Spector.

F RED W. SHERLING Associate Solicitor.

UNITED STATES PATENT OFFICE Certifigate Patent No. 3,709,868 PatentedJanuary 9, 1973 Sidney Spector Application having been made by SidneySpector, the inventor named in the patent above identified, andHofimann-La Roche Inc, Nutley, New Jersey, a corporation of New Jersey,the assignee, for the issuance of a certificate under the provision ofTitle 35 Section 256, of the United States Code, adding the name ofCharles W. Parker as a loint inventor, and a showi and proof of factssatisfying the requirements of the sald section havin been submitted, itis this 5th day of June 1973, certified that the I name of the saidharles W. Parker is hereby added to the said patent as a joint inventorwith the said Sidney Spector.

FRED W. SHERLING Associate Solicitor.

UNITED STATES PATENT OFFICE CERTIFICATE OI CORRECTION Patent No.3,709,868 Dated January 9, 1973 Invent0r($) Sidnev Spector,

It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

Claim 1, Column 8, line 5 "hydroxy" should be hydroxzl At the end of thespecification, described herein was made in part by a co-inventor hereofin the course of work under a grant or award from the Department ofHealth, Education and Welfare.

please insert The invention Signed and sealed this 26th day of February197L (SEAL) Atte st:

EDWARD M.FLETCHER,JR.

C. MARSHALL DANN Attesting Officer Commissioner of Patents FORM PO-IOSO(10-69) uscoMM-oc scan-pen l U. 5 GOVERNMENT PRINTING OFFICE ID.'-S-3l4,

2. The antigen of claim 1 wherein said opium alkaloid derivative is3-0-carboxymethylmorphine.
 3. The antigen of claim 1 wherein saidprotein is bovine serum albumin.
 4. The antigen of claim 3 wherein threeto seven opium alkaloid derivative groups are bonded per molecule ofbovine serum albumin.
 5. An antigen consisting essentially of a carboxylower alkyl derivative of the phenolic hydroxyl group of an opiumalkaloid bonded through a peptide linkage to an amino group of animmunogenic carrier material selected from the group consisting ofproteins and polypeptides.